a fluoride activated methylene blue releasing platform for ... · selected toothpastes were...

1

Electronic Supplementary Information for

A fluoride activated methylene blue releasing platform for imaging

and antimicrobial photodynamic therapy of human dental plaque

Peng Wei,a Fengfeng Xue,a Yunming Shi,b Ross Strand,b Hui Chen,a and Tao Yi*a

a Department of Chemistry, Fudan University, 2005 Songhu Road, Shanghai 200438, China

bP&G Technology (Beijing) Co., Ltd., No. 35 Yuan Road, B zone, Shunyi District, Beijing, 101312,

China

1. Experimental section ............................................................................................................. 2

1.1 Materials and Instruments ............................................................................................... 2

1.2 Synthesis of the compounds ............................................................................................ 2

1.2.1 General procedure for the synthesis of 1-3 ............................................................... 3

1.2.2 General procedure for the synthesis of 4-6 ............................................................... 3

1.2.3 General procedure for the synthesis of FD-F1 ~ FD-F3........................................... 4

1.3 Preparation of probes and analytes .............................................................................. 5

1.4 Preparation of toothpastes ........................................................................................... 5

1.5 Procedure for fluorescence imaging of fluoride uptake in plaque biofilm. ................. 5

1.6 Procedure for PDT in plaque biofilm. ......................................................................... 6

2. Additional Tables .................................................................................................................. 7

3. Additional images and figures ............................................................................................... 8

4. NMR and HRMS spectra of the compounds ....................................................................... 14

5. Reference ............................................................................................................................. 29

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2018

2

1. Experimental section

1.1 Materials and Instruments

All the starting materials were obtained from commercial suppliers and used as

received. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were taken on a

Bruker AV400 nuclear magnetic resonance spectrometer, using DMSO-d6 or CD3CN

as solvent. Proton chemical shifts are reported in parts per million downfield from

tetramethylsilane (TMS), with tetramethylsilane (δ = 0.0 ppm) or the solvent residue

peak CD3CN (1.94 ppm for 1H, 1.32 ppm for 13C), or DMSO-d6 (2.50 ppm for 1H,

39.52 ppm for 13C) as the chemical shift standard. High-resolution mass spectra

(HRMS) were measured on a Bruker Micro TOF II 10257 instrument with

electro-spray ionization (ESI) technique and direct injection method. UV-visible

spectra were recorded on a Shimadzu UV-2550 spectrometer. Steady-state fluorescent

spectra at room temperature were measured on an Edinburgh instrument FLS-920

spectrometer with a Xe lamp as an excitation source.

1.2 Synthesis of the compounds

Scheme S1. Synthesis route of compounds FD-F1 ~ FD-F3

3

1.2.1 General procedure for the synthesis of 1-3

4-Hydroxybenzaldehyde (2.0 g, 16.38 mmol, 1.0 eq) and imidazole (3.35 g, 49.14

mmol, 3.0 eq) were dissolved in 30 mL of dichloromethane, the resulting mixture was

stirred in an ice-water bath. R-Cl (19.66 mmol, 1.2 eq) was dissolved in 10 mL of

dichloromethane and added dropwise. After addition, the mixture was stirred at room

temperature until the reaction completed as indicated by TLC analysis which was

conducted at 1 h intervals. The reaction mixture was poured into 300 mL of ice-water

while stirring, and the resulting mixture was extracted with three 150 mL portions of

dichloromethane. The combined extracts were washed with brine, dried over

anhydrous sodium sulfate and evaporated on a rotary evaporator to afford an oily

residue, which was purified by column chromatography (ethyl acetate/petroleum ether

= 1/20) to yield the pure product 1-3.

4-((tert-butyldiphenylsilyl)oxy)benzaldehyde (1)

White solid. Yield, 5.37 g, 91%. 1H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H),

7.75 – 7.65 (m, 6H), 7.53 – 7.42 (m, 6H), 6.90 (d, J = 8.4 Hz, 2H), 1.05 (s, 9H).

4-((triisopropylsilyl)oxy)benzaldehyde (2)

Colorless oil. Yield, 4.38 g, 96%. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H),

7.84 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 1.34-1.25(m, 3H), 1.07 (d, J = 7.6

Hz, 18H).

4-((tert-butyldimethylsilyl)oxy)benzaldehyde (3)

Colorless oil. Yield, 3.64 g 94%. 1H NMR (400 MHz, DMSO-d6) δ 7.18 (d, J =

8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.05 (t, J = 5.8 Hz, 1H), 4.40 (d, J = 6.0 Hz,

2H), 0.94 (s, 9H), 0.17 (s, 6H).

1.2.2 General procedure for the synthesis of 4-6

To a solution of 1-3 (3.87 mmol, 1.0 eq) in 150 mL of MeOH, cooled with an

ice-water bath, NaBH4 (0.63 g, 16.64 mmol, 1.2 eq) was added in batches. After

addition, the reaction mixture was stirred at this temperature for 30 min and then at

room temperature until the reaction completed as indicated by TLC analysis (typically

within 1-3 h). The reaction mixture was poured into 200 mL of ice-water while

stirring, and the resulting mixture was extracted with three 100 mL portions of

dichloromethane. The combined extracts were washed with brine, dried over

anhydrous sodium sulfate and evaporated on a rotary evaporator to afford an oily

residue, which was purified by column chromatography (ethyl acetate/petroleum ether

= 1/20) to yield the pure product 4-6.

(4-((tert-butyldiphenylsilyl)oxy)phenyl)methanol (4)

White solid. Yield, 1.36 g, 97%. 1H NMR (400 MHz, DMSO-d6) δ 7.69 – 7.63 (m, 5H),

7.47 – 7.42 (m, 5H), 7.07 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.99 (t, J = 5.6 Hz, 1H),

4.33 (d, J = 5.2 Hz, 2H), 1.03 (s, 9H).

(4-((triisopropylsilyl)oxy)phenyl)methanol (5)

Colorless oil. Yield, 1.07 g, 99%. 1H NMR (400 MHz, DMSO-d6) δ 7.18 (d, J =

4


2H), 1.27 – 1.18 (m, 3H), 1.05 (d, J = 8.4 Hz, 18H).

(4-((tert-butyldimethylsilyl)oxy)phenyl)methanol (6)

Colorless oil. Yield, 0.9 g, 98%. 1H NMR (400 MHz, DMSO-d6) δ 7.18 (d, J =


2H), 0.94 (s, 9H), 0.17 (s, 6H).

1.2.3 General procedure for the synthesis of FD-F1 ~ FD-F3

To an aqueous solution (3 mL) of MB (1.11 g, 3.75 mmol, 1.0 eq),

dichloromethane (10 mL) and Na2CO3 (2.38 g, 22.50 mmol, 6.0 eq) were added and

stirred at 60°C under a nitrogen atmosphere. Sodium dithionite (2.61 g, 15.00 mmol,

4.0 eq) was dissolved in 7 mL of water and added dropwise. After addition the

mixture was stirred at 60°C under nitrogen atmosphere until the solution became

yellow (typically within 15-30 min). The mixture was cooled with an ice-water bath,

to which a dichloromethane solution (5 mL) of bis(trichloromethyl)carbonate (1.11 g,

3.75 mmol, 1.0 eq) was added dropwise. After addition, the mixture was stirred for

another 1 h. The dichloromethane layer was separated from the water layer and

quickly dried with anhydrous sodium sulfate. After sodium sulfate was removed by

filtration, the solution was added dropwise to a mixture of 4 - 6 (0.8 eq), DMAP (0.92

g, 7.50 mmol, 2.0 eq), Na2CO3 (1.19 g, 11.25 mmol, 3.0 eq) and 5 mL

dichloromethane. After addition, the mixture was stirred in an ice-water bath for 1 h

and then at room temperature until the reaction was completed as indicated by TLC

analysis.

After the undissolved substance was removed by filtration, the solution was

poured into 200 mL of ice-water while stirring, and the resulting mixture was

extracted with three 100 mL portions of dichloromethane. The combined extracts

were washed with brine, dried over anhydrous sodium sulfate and evaporated on a

rotary evaporator to afford an oily or solid residue, which was purified by column

chromatography (ethyl acetate/n-hexane = 1/10 then 1/5) to yield FD-F1~ FD-F3 as

white solids.

FD-F1. Yield, 0.51 g, 20%. 1H NMR (400 MHz, CD3CN) δ 7.72 (dd, J = 8.0, 1.2

Hz, 4H), 7.49 – 7.45 (m, 2H), 7.42 – 7.39 (m, 4H), 7.26 (d, J = 8.8 Hz, 2H), 7.07 (d, J

= 8.4 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 2.8 Hz, 2H), 6.62 (m, 2H), 5.00 (s,

2H), 2.90 (s, 12H), 1.07 (s, 9H). 13C NMR (100 MHz, CD3CN) δ 156.42, 155.12,

150.11, 136.44, 133.64, 133.56, 131.21, 130.38, 129.05, 128.94, 128.13, 120.58,

120.36, 111.90, 110.86, 68.12, 40.84, 26.89, 19.92. HRMS (ESI), calculated for

C40H44N3O3SSi ([M+H]+) 674.2867, found 674.2871.

FD-F2. Yield, 0.52 g, 23%. 1H NMR (400 MHz, CD3CN) δ 7.30 (d, J = 8.8 Hz,

2H), 7.21 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 2.8 Hz, 2H), 6.64

(m, 2H), 5.07 (s, 2H), 2.90 (s, 12H), 1.31 – 1.22 (m, 3H), 1.09 (d, J = 7.6 Hz, 18H). 13C NMR (100 MHz, CD3CN) δ 156.96, 155.16, 150.07, 133.62, 130.64, 130.21,

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129.02, 128.15, 120.77, 111.88, 110.84, 68.25, 40.83, 18.32, 13.46. HRMS (ESI),

calculated for C33H46N3O3SSi ([M+H]+) 592.3024, found 592.3021.

FD-F3.Yield, 0.37 g, 18%. 1H NMR (400 MHz, CD3CN) δ 7.31 (d, J = 9.2 Hz,

2H), 7.22 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 2.4 Hz, 2H), 6.65

(dd, J = 8.8, 2.8 Hz, 2H), 5.08 (s, 2H), 2.90 (s, 12H), 0.98 (s, 9H), 0.19 (s, 6H). 13C

NMR (100 MHz, CD3CN) δ 150.56, 155.14, 150.06, 133.60, 130.59, 130.44, 128.95,

128.13, 120.97, 118.29, 110.82, 68.21, 40.81, 26.01, 18.58, −4.26. HRMS (ESI),

calculated for C30H40N3O3SSi ([M+H]+) 550.2554, found 550.2567.

1.3 Preparation of probes and analytes

Stock solutions of FD-F1~FD-F3 (1 mM or 2.5 mM for imaging; 5 mg/mL for

aPDT) were prepared in DMSO. The test solution for each probe (10 μM) was

prepared by placing stock solutions in a 10-mL volumetric flask, and diluting with

DMSO-HEPES buffer solution (3/2, v/v, 10 mM HEPES, pH = 7.2). The resulting

solutions were shaken well and incubated at room temperature before recording the

spectra.

Sodium fluoride (NaF) was used as a fluoride source for the fluoride ion sensing

test. The stock solution of NaF was prepared in deionized water. Other analytes were

prepared in ddH2O.

1.4 Preparation of toothpastes

Selected toothpastes were dispersed in 10 mL of distilled water. The obtained

mixture was stirred at 70 °C for 3 h, and then filtered. The filtrate was used for the

analysis of F−. The test solution of FD-F3 (10 μM) to measure F− from different

toothpastes in 10 mM DMSO-HEPES solution (3/2, v/v, 10 mM HEPES, pH = 7.2)

was prepared by placing FD-F3 stock solutions (100 μL) and different toothpaste

stock solutions in 10-mL volumetric flasks, diluting with buffer solution to volume,

and mixing. The resulting solution were shaken well and incubated at room

temperature before recording the spectra.

For comparison, the fluoride concentration was also measured by FISE which is

a type of ion selective electrode that is sensitive to the concentration of F− (detailed

described in U.S. Environmental Protection Agency Website

https://www.epa.gov/sites/production/files/2015-12/documents/9214.pdf).

1.5 Procedure for fluorescence imaging of fluoride uptake in plaque biofilm.

Human subjects (aged 20 to 30 years old) were enrolled in the study according to

the inclusion criteria (Turesky Plaque Index Score 2 to 3). Hydroxyapatite (HA) discs

https://www.epa.gov/sites/production/files/2015-12/documents/9214.pdf

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used as plaque biofilm accumulators were worn by the subjects for 48 hours before

harvesting the natural plaque biofilm. After removal, the HA discs were randomly

assigned to a 2-minute treatment with phosphate-buffered saline (PBS, blank control),

25% slurry of a marketed non-fluoride toothpaste (NFT) or 25% slurry of a marketed

sodium fluoride toothpaste (SFT), and then washed with PBS for three times to

remove the excess toothpaste ingredients.

HA discs were incubated in PBS solution with 0.2%(v/v) DMSO containing 5

µM FD-F3 and 5 µM SYTO® 9 Green Fluorescent Nucleic Acid Stain at 37C for 30

min in the dark, washed with PBS three times to remove the excess probes and bathed

in PBS (2 mL) before imaging.

Confocal laser scanning microscopy (CLSM) images were obtained with

Leica™ TCS SP8 AOBS spectral confocal microscope and Leica™ Confocal

software LAS AF3.3.0 (Leica Mikroskopie GmbH, Wetzlar, Germany). The following

parameters were used: XYZ-scan mode with dual-channel excitation and emission

filters for SYTO® 9 (λex = 488 nm, λ em = 500-550 nm) and FD-F3 (λex = 633 nm,

λem = 670-720 nm), 20x objective lens, and scanning from the biofilm surface to a

total depth of 60 μm with a step size of 5 μm.

1.6 Procedure for PDT in plaque biofilm.

Natural plaque biofilms were obtained according to the above method and

divided into six groups as follows. (1) without any treatment as control; (2) treated

only with free FD-F3 (50 µg/mL) for 30 min; (3) treated only with NaF (50 µg/mL)

for 60 min; (4) treated with FD-F3 (50 µg/mL) for 30 min then NaF (50 µg/mL) for

60 min; (5) treated only with light (100 mW/cm2) for 5 min; (6) treated with FD-F3

(50 µg/mL) for 30 min then NaF (50 µg/mL) for 60 min then light (100 mW/cm2) for

5 min.

The distribution of dead/live bacterial cells in biofilms were observed under

confocal laser scanning microscope (CLSM). Live and dead biofilm bacteria were

simultaneously viewed using the reagents SYTO 9 stain and propidium iodide in the

LIVE/DEAD BacLight Bacterial Viability Kit according to the manufacturer’s

instructions. All data analysis was performed using SPSS 13.0 software package.

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2. Additional Tables

Table S1

Table S1 Other Photophysical Parameters of the probes before and after treated with

NaFa

ɛb (M-1 cm-1) Фc Brightnessd (M-1 cm-1)

FD-F1 (10 µM) 200 - -

FD-F1 (10 µM) + NaF (500 µM)e 6000 0.11 660

FD-F2 (10 µM) 100 - -

FD-F2 (10 µM) + NaF (500 µM)e 2400 0.13 312

FD-F3 (10 µM) 300 - -

FD-F3 (10 µM) + NaF (500 µM)e 49900 0.10 4990

a The data was recorded in DMSO-HEPES solution (3/2, v/v, 10 mM HEPES, pH =

7.2) b Molar absorption coefficient at 667 nm c Ф: absolute fluorescence quantum yield d Brightness = ɛ × Ф, at 667 nm1 e The data was recorded 50 min later after adding NaF

Table S2 The quantitative analysis of different probes (10 μM) after reacted with NaF

(500 μM) for 50 min

MB detection concentration (μM) MB release efficiency (%)

FD-F1 + NaF 4.59 45.9

FD-F2 + NaF 3.69 36.9

FD-F3 + NaF 9.69 96.9

Table S3 Determination of F− content in water with different fluoride sources.

F− source Spiked (μM) Recovered (μM) Recovery (%) RSD

NaF 7 7.4 105.7 0.09

NaF 18 17.1 95 0.03

NaF 31 28 90.3 0.06

SMFP 185 193.3 104.5 0.03

SMFP 570 580.7 101.9 0.05

The data presented has been converted into the concentiation of NaF or SMFP

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3. Additional images and figures

Fig. S1 (a) Fluorescence spectra of FD-F1 (10 μM) before/after treated with 500 μM

NaF; (b) the absorption spectra of FD-F1 (10 μM) before/after treated with 500 μM

NaF (incubated for 50 min, DMSO-HEPES solution. (3/2, v/v, 10 mM HEPES,

pH=7.2), λex =620 nm)

Fig. S2 (a) Fluorescence spectra of FD-F2 (10 μM) before/after treated with 500 μM

NaF; (b) the absorption spectra of FD-F2 (10 μM) before/after treated with 500 μM

NaF. (incubated for 50 min, DMSO-HEPES solution (3/2, v/v, 10 mM HEPES, pH =

7.2), λex = 620 nm)

Fig. S3 Fluorescence intensity changes at 690 nm of probes (50 μM) were incubated in

different cell medium (a) Dulbecco’s modified essential medium (RPMI 1640) and (b)

Dulbecco's Modified Eagle's medium (DMEM) for different time. (The green bar is

640 660 680 700 720 740 760 7800

5

10

15

20

25

FI

( 1

03

a.u

.)

Wavelength (nm)

FD-F1 + NaF

FD-F1

500 550 600 650 700 7500.00

0.02

0.04

0.06

FD-F1

FD-F1 + NaF

Absorb

an

ce

Wavelength (nm)

a b

640 660 680 700 720 740 760 780

0

2

4

6

8

10

FD-F2

FD-F2 + NaF

FI

( 1

02

a.u

.)

Wavelength (nm)500 550 600 650 700 750

0.000

0.005

0.010

0.015

0.020

0.025

FD-F2

FD-F2 + NaF

Ab

so

rba

nce

Wavelength (nm)

a b

FD-F1 FD-F2 FD-F30.0

0.3

0.6

0.9

1.2

1.5

FD-F3 + NaF

Norm

aliz

ed F

I

30 min

12 h

24 h

FD-F1 FD-F2 FD-F30.0

0.3

0.6

0.9

1.2

1.5

FD-F3 + NaF

30 min

12 h

24 h

Norm

aliz

ed F

I

RPMI 1640 DMEM

a b

9

the fluorescence intensity of 10 μM FD-F3 and 20 μM NaF reacted for 50 min as

reference)

0 50 100 150 200 2500

5

10

15

20

25

FI

( 1

03

a.u

.) FD-F1

Time (min)

FD-F2

Fig. S4 Fluorescence intensity enhancement at 690 nm for FD-F1 (10 μM) and

FD-F2 (10 μM) with time increase after treated with 500 μM NaF. (DMSO-HEPES

solution (3/2, v/v, 10 mM HEPES, pH = 7.2), λex = 620 nm)

Fig. S5 HRMS analysis of compound FD-F1 after reacted with NaF (the theoretical

molecular mass of the MB is 284.1216, [M – Cl−]+ )

10





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Fig. S8 HPLC analysis of MB (10 μM) and different probes (10 μM) after reacted

with NaF (500 μM) for 50 min.

500 550 600 650 700 7500.0

0.1

0.2

0.3

0.4

0.5

Blank and

with other anions

With NaF

Ab

so

rba

nce

Wavelength (nm)

Fig. S9 The absorption spectra of probe FD-F3 (10 μM) when treated with 500 μM

various tested analytes (incubated for 50 min, DMSO-HEPES solution (3/2, v/v, 10

mM HEPES, pH=7.2), λex=620nm)

7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0

FD-F3 + NaF

FD-F2 + NaF

Time (min)

FD-F1 + NaF

MBⅳ

ⅰ

ⅱ

ⅲ

12

2 4 6 8 10 120

5

10

15

20

25

FI

( 1

03

a.u

.)FD-F3 only

With 500 M NaF

pH

Fig. S10 Fluorescence intensity at 690 nm at different pH levels before and after

treatment with 500 μM NaF for 50 min. (DMSO-HEPES solution, 3/2, v/v, 10 mM

HEPES, pH = 7.2; λex = 620 nm)

Fig. S11 Fluorescence intensity of FD-F3 (10 μM) at 690 nm (a) with different

incubation times after treatment with 500 μM SMFP; (b) with different incubation

times after treatment with 500 μM SMFP and incubated for 30 min after treatment

with 500 μM NaF. (c) Fluorescence spectra of FD-F3 after adding 500 μM different

inorganic fluoride. (d) Fluorescence intensity of FD-F3 with different incubation time

after treated with 500 μM SMFP and TBAF. (DMSO-HEPES solution, 3/2, v/v, 10

mM HEPES, pH = 7.2; λex = 620 nm)

0 2 4 6 8 10 120

2

4

6

8

10

FI

( 1

03

a.u

.)

FD-F3 + Na2FPO3

Time (10 min)

0

4

8

12

16

20

FI

( 1

03

a.u

.)

Time (minute)

TBAF

SMFP

30 60 90 120

6 4 0

6 6 0

6 8 0

700

720

740

760

780

0

5000

10000

15000

20000

25000

30000

KFN

aFCsF

Wavelength (nm

) Flu

ore

scen

ce In

ten

sit

y (

a.u

.)

0

2

4

6

8

10

FI

( 1

03

a.u

.)

NaF-30 min

SM

FP

-120 m

in

SM

FP

-60 m

inFD

-F3 o

nly

SM

FP

-30 m

in

a

c

b

d

13

0 200 400 600 800 1000

0

2

4

6

8

10

12

FI ( 1

03

a.u

.) y = 232.14286 + 11.03771x

R2 = 0.98566

Na2FPO3 (M)

Fig. S12 Fluorescence intensity of FD-F3 (10 μM) at 690 nm with different

concentrations of SMFP incubated for 50 min. (DMSO-HEPES solution, 3/2, v/v, 10

mM HEPES, pH = 7.2; λex = 620nm)

A B C D E F-10

0

10

20

30

40

(Dead b

acte

ria -

basic

)/basic

1

00%

Fig. S13 Proportion of dead bacteria after treated with (A) either no drug as control;

(B) treated only with free FD-F3 (50 µg/mL) for 30 min; (C) treated only with NaF

(50 µg/mL) for 60 min; (D) treated with FD-F3 (50 µg/mL) for 30 min then NaF (50

µg/mL) for 60 min (E) treated with FD-F3 (50 µg/mL) for 30 min then NaF (50

µg/mL) for 60 min then light (100 mW/cm2) for 5 min; (F) treated only with light

(100 mW/cm2) for 5 min. ( The light was using laser at 655 nm. )

14

4. NMR and HRMS spectra of the compounds

Fig. S14 1H-NMR of compound 1 in DMSO-d6

15


16


17


18


19


20

Fig. S20 1H-NMR of compound FD-F1 in CD3CN

21

Fig. S21 13C-NMR of compound FD-F1 in CD3CN

22

Fig. S22 HRMS of compound FD-F1

23


24


25


26


27


28


29

5. Reference

1. Y. Zhang, S. Swaminathan, S. Tang, J. Garcia-Amorós, M. Boulina, B. Captain, J.

D. Baker and F. M. Raymo, J. Am. Chem. Soc., 2015, 137, 4709-4719.

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